Univ Of Massachusetts Med Sch Worcester

NIH Research Projects · FY 2025 · 2025-05

Project Summary Advanced age is a single risk factor for developing serious complications from infections with respiratory viruses such as influenza, SARS-CoV-2 or RSV. These complications are reflected in thrombotic outcomes including microthrombosis, myocardial infarction and pulmonary embolism. The actual cause and mechanisms of these thrombotic outcomes remains elusive. Pathogen spreading and crossover of these viruses into the circulation is regulated by various mechanisms, some of which involve lytic programmed cell death pathways such as necroptosis executed by membrane channels formed by oligomerized phosho-Mixed Lineage Kinase Domain Like Pseudokinase (MLKL). Platelets express MLKL, have a plethora of immune-sensing viral receptors and are the major blood component responsible for thrombotic outcomes. We have shown that respiratory viral RNA can be found in circulating platelets from influenza patients and preliminary result support channel formation. In this proposal, we hypothesize that platelet-pMLKL channel formation, mediated by influenza, leads to cytoplasmic S100 content release and contributes to immunothrombosis with age. We propose to test this hypothesis with the following aims: 1. Determine the MLKL-specific platelet content release and whether platelets undergo necroptosis as a result of influenza and (or) age, and 2. Determine the contribution of age to MLKL-activation and immunothrombotic aggregates during infection. The proposed studies are central to elucidating mechanisms that may increase immunothrombotic risk and adverse cardiovascular outcomes beyond classical platelet activation, with advanced age, and provide a basis for novel and targeted treatments for prevention.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY/ABSTRACT Alcohol Use Disorder (AUD) is a chronic, relapsing disease, affecting 10.6% of United States adults. In the treatment seeking population, periods of abstinence define a highly vulnerable portion of the AUD cycle, as during this time individuals report heightened craving and experience increased negative affect. Given the heterogeneity of alcohol use and behaviors observed in the clinical population, animal models highlighting individual differences may provide novel insight to the development of targeted therapeutic interventions. We propose the use of an operant conditioning task known as Structured Tracking of Alcohol Reinforcement (STAR) to define subpopulations of mice displaying high, low, or compulsive (aversion resistant) ethanol intake behaviors. Our data suggest that mice displaying compulsive drinking may represent a particularly vulnerable subpopulation, as these mice display greater ethanol seeking in protracted abstinence. Our data recoding calcium transients during this ethanol seeking session implicate the bed nucleus of the stria terminalis (BNST) as a potential driver of maladaptive behavior in the compulsive phenotype. Part of the extended amygdala abstinence network, the BNST is a critical brain region for the integration of negative affect and alcohol-related behaviors. However, the BNST is highly heterogenous, composed of specialized subpopulations of neurons. One largely unstudied population of BNST neurons are the protein kinase C delta (PKCδ)-expressing neurons. Despite strong support for central amygdala (CeA) PKCδ neurons in both aversion-resistant ethanol intake and reinstatement of ethanol-seeking behaviors, no studies have examined BNST PKCδ neurons in the context of ethanol. However, our lab recently demonstrated a direct projection from CeA PKCδ neurons to BNST PKCδ neurons, and preliminary data demonstrate engagement of BNST PKCδ neurons during an ethanol drinking bout as well as changes in activity following cycles of ethanol exposure and withdrawal. Altogether these data support BNST PKCδ neurons as a novel circuit component of compulsive ethanol intake. These findings led us to develop our central hypothesis that abstinence from ethanol exposure induces changes in BNST PKCδ neuron activity and their associated circuitry to drive compulsive ethanol intake. To test this hypothesis, the mentored (K99) phase will provide training in in vivo fiber photometry to evaluate adaptions in BNST PKCδ calcium transients across an abstinence period during operant behavior (Aim 1) and ex vivo slice wide-field calcium imaging to dissect population dynamics in BNST PKCδ calcium transients in response to ethanol exposure (Aim 2). The independent (R00) phase will evaluate adaptations in BNST PKCδ neuron afferents, exploring the ability of dysregulation in these areas to modulate compulsive intake through their influence on the BNST (Aim 3). These proposed studies and related career development training plan in this Pathway to Independence Award collectively provide the ideal mechanism to transition to a career as an independent addiction neuroscientist.

NIH Research Projects · FY 2026 · 2025-05

Project Abstract The overall goal of this proposal is to gain a new understanding of the factors that contribute to chemoresistance in BRCA1 and BRCA2 (BRCA) mutant hereditary breast and ovarian cancers (HBOC). Currently clinical strategies rely on chemotherapies and poly (ADP-ribose) polymerase inhibitors to control malignant disease. Unfortunately, tumor chemoresistance frequently occurs which necessitates studies that uncover the critical factors leading to chemoresistance. We have recently discovered chemoresistance is linked the single-stranded (ss)DNA gap suppression in multiple models of HBOCs and patient tumors. Our work has generated a paradigm shift that ssDNA predicts sensitivity whereas gap suppression predicts resistance. To expand upon our model that ssDNA gaps are the sensitizing lesions in BRCA cancers, I propose two aims. In Aim 1, I will determine if an axis of chemoresistance in BRCA1 deficient cancers, linked to restored homologous recombination, is instead mediated by gap suppression. In Aim 2, I will determine if gap suppression in a chemoresistant BRCA2 tumor model is linked to the activation of translesion synthesis and how translesion synthesis can be overcome to resensitize these cancers. Together, these aims increase our knowledge of the basic factors that lead to chemoresistance in the clinic and will provide new insights into the vulnerabilities unique to BRCA deficient cancers. Moreover, by identifying unique factors contributing to chemoresistance, we will develop an understanding for potential druggable targets and biomarkers for future personalized chemotherapy. Existing chemotherapies are constrained by their side-effect profiles, often taxing patients' tolerance and potentially inducing secondary malignancies. Generating a new understanding of the factors contributing to chemoresistance is pertinent to improving patient outcomes.

NIH Research Projects · FY 2026 · 2025-05

Abstract Familial platelet disorder (FPD) is a rare, inherited, dominant disease that is associated with high lifetime-risk of developing hematologic malignancies. FPD patients can live for decades in a “pre-leukemic” period before disease onset, opening a therapeutic opportunity for the development of preventive treatments. Molecularly, FPD is caused by germline mutations in the RUNX1 gene that reduce RUNX1 function, and is associated with clonal hematopoiesis and deregulated cytokine production in the immune system. Our preliminary results show that germline Runx1 mutations in mice create a pre-leukemic bone marrow niche marked by increased inflammatory cytokines, reduced DNA damage repair (DDR) response, and predisposition to hematologic malignancies. Furthermore, we found that treatment with the tyrosine kinase imatinib restored DDR response in vitro. Based on these preliminary data, we hypothesize that RUNX1 mutations hamper DDR response in hematopoietic stem and progenitor cells thereby promoting clonal hematopoiesis and the risk to developing hematologic malignancies. Furthermore, we propose that treatment with the tyrosine kinase inhibitor imatinib reduces clonal selection and may prevent disease onset by restoring DDR response. This hypothesis will be tested in the following three Specific Aims: 1. Determine the efficacy of the tyrosine kinase imatinib in restoring DDR response in human FPD pre-malignant cells. 2. Identify and characterize the pathways targeted by imatinib treatment in DDR of RUNX1-mutant hematopoietic cells. 3. Study the efficacy of tyrosine kinase inhibitors in preventing FPD-associated hematologic malignancies in mice. Together, these studies will provide mechanistic and functional insights into the role of RUNX1 mutations in DDR pathways, clonal selection and predisposition to hematologic malignances. Furthermore, they will establish whether treatment with tyrosine kinase inhibitors can reduce clonal selection and delay the onset of hematologic malignancies. These results may have a direct impact on the initiation of a phase-II clinical trial for FPD patients.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Genome maintenance is crucial to cellular and organismal health. In conditions where DNA damage repair (DDR) pathways are disrupted, patients develop severe multisystem disorders. While these diseases impact distinct repair pathways, they share some phenotypic overlap, such as premature aging and neurodegeneration. This observation suggests that genome instability promotes aging and neuronal death. One such DDR disease is Ataxia Telangiectasia (A-T), a life-limiting DDR disease characterized by cerebellar degeneration, immunodeficiency, and susceptibility to cancer. A-T is caused by biallelic loss-of-function (LoF) mutations at the A-T mutated (ATM) locus, which encodes ATM kinase. A-T relevant mutations are thought to interfere with one or more of its functions, most prominently ATM’s role orchestrating DDR and sensing reactive oxygen species. Loss of these functions results in increased DNA damage and higher ROS in A-T patient cells. Ataxia in A-T coincides with loss of cerebellar volume while sparing other brain regions. Histology shows that Purkinje cells (PCs), the sole output of the cerebellar cortex, and cerebellar granule neurons die over the course of A-T. Our preliminary data shows that deletion burden is higher in A-T neurons from the pre- frontal cortex, but mutation burden analysis is lacking from disease-affected cell types. How ATM LoF impacts PC genome stability, and consequently PC function and longevity, remains unknown. I propose to characterize how ATM LoF impacts the PC genome and transcriptome at the single-cell level in the human cerebellum. Post-mortem human brain was chosen over animal models because A-T mammalian models fail to phenocopy human cerebellar pathology. This experiment requires a single-cell analysis because PCs are very rare, comprising <1% of the cells of the cerebellum, so a “bulk” analysis would obscure PC- specific patterns. A pure population of PCs is a prerequisite for single-cell somatic mutation analysis. However, a robust protocol to isolate rare PCs for single-cell molecular analysis was lacking before I started this project. To circumvent this hurdle, I adapted a technique to isolate soma from fresh frozen brain. I applied PC soma to a new muti-omic protocol capable of capturing DNA and RNA from the same single cell to human brain for the first time. Aim 1 will compare single cell DNA mutation burden between PC from A-T and matched controls. Aim 2 will synthesize mutation characteristics – context, distribution, size (single base variants, small/large insertion or deletions, structural variants) – into signatures. Signatures will suggest mechanisms. Aim 3 will investigate the connection between DNA damage and transcriptional changes, linking genotype and phenotype. Further, this work will provide the fellow with exemplary training in genomics, neurobiology, statistics, and bioinformatics.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY Common nerve injuries often lead to long-term disabilities in the central nervous system and in the peripheral nervous system with increased adult age. Understanding how axon guidance is regulated in the mature nervous system is critical to understanding how the nervous system changes after development and to developing therapies aimed at reestablishing proper neural circuitry across lesions. Caenorhabditis elegans is a highly tractable model to dissect conserved molecular and cellular mechanisms that regulate axon regeneration and axon guidance. With this system, I have identified an extracellular matrix protein that guides regenerating motor axons specifically in the post-development nervous system and is required for functional recovery. I will apply my developing skills in detailed genetic analyses, laser axotomy, in vivo imaging, sequencing, and bioinformatics to further determine the post-developmental specific role of this protein in axon guidance. In addition to providing a critical advance in understanding the fundamental mechanisms of axon guidance, it will also inform future therapeutic approaches that can be manipulated to promote targeted axon guidance in the injured adult nervous system.

NIH Research Projects · FY 2026 · 2025-04

Project Summary Mutations in isocitrate dehydrogenase 1 (mIDH1) are prevalent in various solid tumors, including intrahepatic cholangiocarcinoma (ICC), leading to the production of (R)-2-hydroxyglutarate (2HG). This oncometabolite disrupts epigenetic and metabolic processes by inhibiting enzymes involved in DNA demethylation. I have developed a genetically engineered mouse model (GEMM) that recapitulates the genetics and histopathologic features of human mIDH1 ICC. I revealed that mIDH1 creates an immunosuppressive microenvironment in ICC centered on dual 2HG-mediated mechanisms suppressing CD8+ T cell anti-tumor activity. These properties are reversed by mIDH1 inhibition leading to reduced tumor growth associated with pronounced activation of immune stimulatory interferon (IFN) signaling and CD8+ T cell recruitment and effector function. My progress reveals that restoring antitumor immunity is central to mIDH1 inhibitor efficacy. However, we still have an incomplete understanding of the basis of mIDH1-mediated immune evasion that must be addressed to advance my mechanistic understanding and guide therapeutic development. My preliminary data suggest that mIDH1 ICCs are 'immunologically cold' due to two distinct mechanisms: a tumor cell-intrinsic pathway that hampers immunological recognition of the tumor cells and a paracrine program that curbs immune cell function. i) mIDH1 suppresses innate immunity: My research shows mIDH1 suppresses innate immunity, as evidenced by a low type I IFN gene signature and silencing of the cGAS sensor. Conversely, AG120 rapidly induces endogenous retrovirus (ERV) expression. Notably, cGAS and ERV-derived reverse transcriptase deletion blocks AG120 efficacy. ii) Metabolic crosstalk: My recent studies show that mIDH1 also impairs immune cell function via metabolic crosstalk by affecting T cell effector function and macrophage polarization. My preliminary studies suggest that these effects are due, in part, to the uptake of tumor-derived 2HG by these immune lineages as well as by metabolic cross-competition, with mIDH1 driving high levels of glycolysis, potentially limiting nutrient availability required for immune cell function. I aim to test whether these two processes collectively cause the immune suppressive phenotype of mIDH1 tumors. This study will elucidate the biology of this oncogene, and my data indicate that mIDH1 gliomas share common themes, suggesting a broader impact. I will focus on three specific aims: 1) Decipher the role of viral mimicry in response to mIDH1 inhibition; 2) Explore the contribution of metabolic crosstalk to immune evasion. These studies will offer novel insights into the mechanisms of mIDH1 inhibition in cancers, potentially leading to the development of more effective therapeutic strategies and enhancing understanding of tumor-immune communication. To support this work, I have outlined a career development plan to refine skills in laboratory management, knowledge dissemination, and securing independent funding. Securing a K22 award will be instrumental in achieving my long-term goal of becoming an independent researcher in cancer metabolism and immunology and will propel my pursuit of innovative cancer therapies.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY We propose to create a knowledgebase to extract, accumulate, organize, annotate, and link growing bodies of information related to the Osteoarthritis Initiative (OAI), one of the most prolific osteoarthritis (OA) cohorts in history. The OAI is a unique and rich 16-year multicenter observational study with comprehensive publicly available clinical data, medical images, genetics, and biospecimens from 4,796 people with or at risk for knee OA. Consequently, the OAI is a valued resource to a large and dynamic research community, with ~1,000 publications (>100/year for the past 6 years) and >770 researchers having multiple publications using OAI data. Despite the OAI’s widespread adoption among OA researchers, it retains considerable untapped potential to support investigations in wide-ranging OA-related fields. Utilization of OAI data, images, and biospecimens would be enhanced by a centralized resource that leverages contemporary technology to raise awareness in a broader research community and provide logistical support in leveraging the OAI, ensuring that the OAI will be a durable resource. We propose to create a biomedical knowledgebase to promote and facilitate the use of the OAI to accelerate discovery. We will use trustworthy governance and good data practices to offer high-quality and efficient services to meet the community’s needs using an intuitive and centralized resource. We will create two broad platforms with the first being the Online OAI Resource Library, primarily a stand-alone online resource with 1) foundational documents (OAI Archive; e.g., approved consent forms), 2) presentations and educational opportunities (OAI.Edu), 3) annual State of the OAI Reports, 4) an online calculator for descriptive analyses (OAI Explorer), 5) a database of available OAI biospecimen, and 6) datasets that instructors can use in classes (OAI Learning Lab). Our second will be the OAI Engagement Services, where the OAI CORE will directly engage with interested investigators to assist with an array of activities, including 1) discussing available data and limitations, 2) creating parsimonious data or imaging sets, 3) preparing new datasets to link to the publicly available data, and 4) connecting new OAI investigators with experienced OAI investigators during one-on-one and group sessions – a catalyst to facilitate collaboration and new research. We will also monitor and report >25 metrics of usage, utility, and scientific impact and advancement of knowledge and understanding. This knowledgebase will promote and maintain the OAI, facilitate high-quality studies of existing data, and encourage the generation and sharing of new data from the OAI among OA researchers and the broader research community, accelerating the pace of discoveries.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlapping genetic and pathological features. While neuronal degeneration predicts the clinical and pathological manifestations of these disorders, non-neuronal cells significantly contribute to disease pathogenesis. Indeed, alterations in immune response and dysregulation of microglia are early features of ALS and FTD, and are believed to contribute to disease progression. A prominent, pathological feature of ALS, FTD and related neurodegenerative diseases is TAR DNA binding protein 43 (TDP-43) dysfunction. TDP-43 is an RNA/DNA-binding protein with many roles in RNA and DNA processing, yet the role of TDP-43 in these neurodegenerative diseases remains poorly defined. We have been investigating the link between traumatic brain injury (TBI) and neurodegeneration, as TBI is a known risk-factor for developing neurological disorders with TDP-43 pathology. Using a knock-in mouse model of mutant TDP-43 (i.e., ALS/FTD-TDP mice), we conducted neurological and omics analyses in ALS/FTD-TDP versus WT mice following a mild, concussive TBI. We found that ALS/FTD-TDP mice are more susceptible to neurological deficits following TBI compared to their WT counterparts, a phenotype that was accompanied by changes in innate immune pathways involved in nucleic acid sensing in ALS/FTD-TDP mice. Microglia are key modulators of innate immunity in the CNS, and are critical for recovery after injury. Therefore, Aim 1 in this proposal will probe innate immune pathways and characterize CNS cell types, including microglia subtypes, within ALS/FTD-TDP mice both at baseline and after TBI. To investigate the mechanistic impact of TDP-43 dysfunction on the properties of microglia, we will also employ a human induced pluripotent stem cell (iPSC) derived microglia (iMG) model in Aim 2. Complementary to our ALS/FTD-TDP mouse model, iMGs will allow us to determine the effects of TDP-43 mutation or TDP-43 knock-down on microglial function. We will further utilize this iMG model through xenotransplantation of iMGs into the mouse brain, allowing us to elucidate the impact of TDP-43 mutation specifically in microglia in vivo. The outcomes of this proposal have the potential to uncover mechanisms by which TDP-43 mis-expression alters the innate immune landscape in the CNS, including under conditions of CNS insult.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY/ABSTRACT Caspases are critical initiators and executioners of programmed cell death (PCD) pathways. While these pathways function in antiviral defense, little is known about the mechanisms by which caspases and PCD pathways restrict virus replication. This is particularly true in mucosal barrier tissues, such as the oral cavity which is the entry site of numerous human pathogens including herpes simplex virus 1 (HSV-1). Recently, we found that oral epithelial keratinocytes undergo a pro-inflammatory cell death process known as pyroptosis when treated with activators of the tumor necrosis factor receptor (TNFR)-dependent extrinsic PCD pathway. In addition, activation of this extrinsic PCD pathway inhibited the replication of a HSV-1 mutant lacking the viral protein ICP6 (HSV-1 ∆ICP6), which has previously been shown to be unable to inhibit extrinsic cell death in other cell types. TNFR-mediated restriction of HSV-1 ∆ICP6 in keratinocytes was blocked with the addition of a pan-caspase inhibitor, implicating a role for caspases in inhibiting this viral mutant. Thus, oral keratinocytes represent a robust biologically relevant system to study the mechanisms by which caspases restrict virus replication. We found that several caspases, including caspase-3 and -8, were activated in normal oral keratinocytes where the TNFR-dependent extrinsic PCD pathway was activated. Further, we found that restriction of HSV-1 ∆ICP6 under these conditions occurs at a late step in the viral replication cycle (e.g., egress). Additional investigation revealed that caspase-3 activation in oral keratinocytes promotes cleavage of a pool of vacuolar protein sorting 4A (VPS4A), a component of the endosomal sorting complex required for transport (ESCRT)-III pathway. VPS4A is an AAA-ATPase known to function in HSV-1 egress. Thus, the central hypothesis of this proposal is that extrinsic cell death activation results in caspase-3-mediated cleavage of VPS4A, resulting in the formation of a dominant negative protein that inhibits HSV-1 egress. We will investigate this hypothesis with two distinct aims. In Aim 1, we will define the mechanism and functional consequences of VPS4A cleavage on HSV-1 replication. In Aim 2, we will determine the mechanism by which TNFR-mediated extrinsic cell death activation restricts HSV-1 ∆ICP6. Completion of these aims will lead to a better understanding of the molecular mechanisms by which caspases restrict HSV-1 and fill an important gap in the field regarding how these proteases function in antiviral defense at barrier tissues.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY One-time in vivo gene therapies, based on adeno-associated viral (AAV) vector delivery of genes encoding therapeutic proteins, noncoding RNAs, or genome/epigenome editors, may provide long-lasting (years of, or even lifelong) treatments or cures for many rare and common diseases. However, we cannot currently tune or inactivate transgene expression to reflect disease progression or the emergence of adverse events or contraindications over time, limiting the utility of AAV gene therapies. The constrained packaging capacity of AAV vectors (~4.7 kb) has thwarted development of genetic switches that can regulate transgene expression timing or levels—the solution to these limitations. To date, just a few AAV-compatible switches function in animals. Typically, these switches are activated or repressed by ligands with undesirable side effect, including rapamycin, a potent immune suppressant; tetracycline, an antibiotic not suitable for chronic use; and branaplam, a compound that causes peripheral neurotoxicity. We previously engineered an efficient synthetic RNA ON switch based on our novel self-cleaving ribozyme T3H38, which is regulated by a complementary morpholino oligonucleotide. At 63 bp long, the tiny T3H38 ribozyme can be inserted into the 3′ UTR of a transgene. Its regulator, a 25 nt morpholino oligo is part of a class of chemically modified RNA drugs that have proven safe for chronic use in humans. The T3H38 ribozyme showed a ~200-fold regulation in AAV transgene expression in mice upon administration of the complementary morpholino oligo to the animals. Optimizing the T3H38 ribozyme switch towards a leak-free system with no detectable baseline transgene expression in the absence of the morpholino oligo could transform AAV-based gene therapy. For example, it can enable safe use of AAV to express a variety of transgenes with narrow therapeutic windows (e.g., cytokines, hormones, genome editors), where an overdose or prolonged exposure can cause severe adverse events, or to conditionally express therapeutics with major contraindications (e.g., cytokines), specific disease conditions where a drug should not be used or should be discontinued. In preliminary studies, we have developed an enhanced RNA ON switch (regulatory range: up to 35,000-fold in mice) based on the T3H38 ribozyme. Here, we will (i) optimize the enhanced RNA switch to engineer an ultra-efficient switch with negligible leakiness; (ii) use the optimized switch for precise dose control of an AAV expressing erythropoietin—a paradigmatic biologic with a short half-life, a narrow therapeutic window, and major contraindications—for the treatment of chronic Epo-deficient anemia in a mouse model; and (iii) further optimize the switch system in mouse airways for temporal control of an AAV expressing a broadly anti-coronavirus immunoadhesin—a prototype of a broadly effective prophylaxis for immunocompromised individuals against a panel of coronaviruses of pandemic potential. The completion of this project will provide a broadly useful and potentially transformative regulatable gene therapy technology and proofs of concept showcasing the utility of this technology.

NIH Research Projects · FY 2026 · 2025-02

Abstract The immune system has evolved mechanisms to recognize and respond to cell injury and this response contributes in important ways to both health and disease. In this process, alarm signals called Damage Associated Molecular Patterns (DAMPs) are released from injured cells and these are then detected by receptors on innate immune cells, which trigger sterile inflammation. These responses are thought to be important for host defense, but also cause tissue damage that contributes to a number of diseases. Because of this, it is important to identify the key DAMPs that drive these responses and also the receptors that engage these ligands and mediate their effects. While we and others have discovered a few DAMPs/DAMP receptors, it is clear that there are others yet to be discovered. Moreover, the DAMP/DAMP receptors have been primarily studied in mice and less is known about these in humans. This grant is based on our preliminary discoveries of a novel DAMP receptor in humans, Clec17a, a related murine counterpart, CD209f/g, and their novel DAMP ligand, GAPDH, which is highly conserved, broadly expressed, and abundant. These discoveries form the basis for this grant. Our central hypothesis is that Clec17a in humans and CD209f/g in mice are key receptors on innate immune cells that sense the release of GAPDH from injured cells and help drive the ensuing sterile inflammatory response and its associated pathology. We have 3 specific Aims to elucidate the structure and function of this novel DAMP and its receptors. Aim 1 will elucidate in humans what innate immune cells are stimulated by GAPDH, the nature and biology of their responses and the role of Clec17a in this process. In addition, this aim will seek to determine the specificity of this response and how it fits in with responses stimulated by other DAMPs. The importance of these goals is that they will establish a novel DAMP-DAMP receptor in humans, elucidate their pathophysiology, and provide insight into how humans recognize and respond to cell injury. Aim 2 will investigate the structural and functional murine counterpart of human Clec17a, and elucidate its role, and that of GAPDH, in mice. Aim 3 will elucidate the underlying molecular mechanisms by which human Clec17a and mouse CD209f/g trigger innate immune cells and ultimately drive host defense and pathobiology.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY The innate immune response is highly regulated and the precise timing of gene regulation is crucial for cellular survival. A primary feature of initial innate immune response cascades is the timely mounting of transcriptional and processing steps that underlie immune gene expression, with extensive rewiring of mRNA levels and compositions. Thus, the rates at which mature mRNAs are produced during immune responses might be a critical regulatory step. Published and preliminary studies suggest gene-specific kinetic regulation of mRNA biogenesis may underlie changes in gene expression levels and transcriptome complexity in the early innate immune response. Yet, it remains unclear how the temporal coordination between transcriptional and co-transcriptional processes governs the timing of immune gene expression and if this co-regulation underlies the ability to mount a productive immune response upon first encounter with a stimulus. Gene-specific co-regulation of mRNA biogenesis rates may also allow for efficient immune responses after repeated stimulation. Some innate immune cells, such as macrophages, undergo epigenetic reprogramming following initial exposure to immunogenic signals to mount long-term memory, a phenomenon called trained immunity. Trained macrophages exhibit a faster, more robust innate immune response to resist reinfection, suggesting that epigenetic changes may enable gene-specific kinetic regulation of mRNA biogenesis. How the kinetics of transcription and splicing are regulated by epigenetic changes during immune responses is unknown. This project seeks to uncover mechanistic insights into the regulation and impact of stimulus-induced immune mRNA biogenesis. This work will test the hypothesis that cellular immune responses are driven by a series of regulated changes in the kinetics of transcriptional and co-transcriptional processes, ultimately determining gene expression dynamics over response time. Aim 1 will examine coordination of transcription and splicing kinetics using lipopolysaccharide (LPS)-stimulated human monocyte-derived macrophages (MDMs). Simultaneous sequencing of nascent and mature RNA at non-overlapping 15-minute intervals over a 2-hour stimulation time course will be used to characterize how transcription and splicing rates govern the timing of early immune gene expression. The functional output of key cytokines will be measured in cell supernatants over the time course. Aim 2 will explore how trained immunity-associated epigenetic rewiring impacts transcriptional and co- transcriptional kinetics in MDMs. Trained and un-trained MDMs will be stimulated with LPS in a 2-hour time course as in Aim 1 to assess transcription and splicing rates, then harvested to assess chromatin accessibility. Cytokine output will also be assessed over time, as in Aim 1. Overall, this work will elucidate the mechanisms by which epigenetic rewiring drives resistance to reinfection in trained immunity, illuminate how kinetic regulation of transcriptional and co-transcriptional processes contributes to innate immune phenotypes, and provide the fellow with training in RNA biology, innate immune signaling, computational biology, and immunogenomics.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY This project aims to systematically investigate a critical, but poorly understood aspect of drug resistance evolution: the interdependence of mutations that disrupt drug binding (usually also decreasing enzyme activity) and compensatory mutations that increase enzyme activity. Combinations of these types of mutations are typically observed in pathogens that evolve clinically relevant resistance. The mechanisms that underlie these mutations have not been extensively investigated. Here, we plan to comprehensively analyze all combinations of mutations in Mpro from SARS-CoV-2 that disrupt binding to nirmatrelvir with those that increase enzyme activity. Nirmatrelvir is the active component in Paxlovid that is currently an effective treatment for COVID19. We developed a yeast screen for Mpro activity that is both safe because it does not create or use virus and biologically relevant because it uses a cut-site that is used by the virus. Mutations we identified with this screen have been observed in SARS-CoV-2 viruses selected for resistance, further indicating the screens biological relevance. In the first aim of this work, we will quantify how all combinations of drug-binding and increased activity mutations impact Mpro activity and drug disruption in our yeast screen. The resulting data will be analyzed to elucidate patterns and their structural underpinnings. As compensatory mutations can be specific, in the second aim, we will perform an unbiased analysis of all possible point mutations in the background of two mutations that strongly disrupt nirmatrelvir binding. Together these aims will provide a new view of how mutational interdependencies impact the evolution of drug resistance in a clinically important pathogen.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Characterization of HIV reservoirs and the immunoevasion mechanisms permitting persistence has largely focused on CD4+ T cells. Yet macrophages represent a physiologically relevant reservoir, with characteristics that may uniquely contribute to viral persistence. They can bud and store virus within virus-containing compartments, which largely exclude neutralizing antibodies, and resist the cytopathic effects of infection. Furthermore, en masse, macrophages vs CD4+ T cells are more resistant to killing by CTL and NK cells. Our published work shows that macrophages resist both NK cell innate cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), but the mechanisms of this evasion are unknown. As passive transfer of broadly neutralizing antibodies has shown much promise in the cART-free treatment of infection, understanding how macrophages respond to HIV antibodies and how they might succumb to ADCC would aid the design of inclusive HIV cure strategies. Interestingly, while HIV antibody-enhanced NK cell signaling/activation and release of perforin/granzymes towards infected macrophages vs CD4+ T cells is significantly lower, HIV-specific CAR T cells activate in response to both infected cell types equally well. The overall objective of this research project is to determine the mechanisms of how infected macrophages antagonize NK cell signaling that induces ADCC. Given that NK cells signal in response to multiple target cell surface ligands, which are likely different on CD4+ T cells vs macrophages, the central hypothesis of this project is that infected macrophages present an inhibitory signal that is not on CD4+ T cells, which counteracts NK cell ADCC. Specific Aim #1 will characterize the effects of HIV accessory proteins on ADCC-triggered NK cell signaling towards CD4+ T cells vs macrophages. Previous studies suggest that NK ligands can affect ADCC. Since the HIV accessory proteins Nef and Vpu differentially affect CD4+ T cells and macrophages, they may also elicit differential regulation of NK cell ligands in these cell types, leading to differences in ADCC-triggered NK cell signaling. Using HIV with genetically ablated accessory proteins, we propose to assess how each protein regulates the NK cell ligand repertoire and ADCC-triggered NK cell signaling. While these viral proteins may explain the ADCC differences towards the targets, Specific Aim #2 will define how Fc𝛾 receptor (Fc𝛾R) binding to HIV antibody, which also binds to the surface Env on macrophages (in cis) affects ADCC-triggered NK cell signaling (in trans). Fc𝛾R expression on targets cells, which inhibits ADCC towards transplanted cell therapeutics, may be the dominant factor that protects macrophages. We propose to use CRISPR KO of Fc𝛾Rs in macrophages and lentiviral overexpression of Fc𝛾Rs in CD4+ T cells to determine the effects of Fc𝛾Rs on ADCC towards infected cells and uninfected targets that overexpress HIV Env. Together, these studies will provide insight into the mechanisms of macrophage resistance to ADCC, which may help in the design of more effective and inclusive HIV cure strategies, such as blocking accessory protein function with small molecules or design of bNAbs that limit macrophage Fc𝛾R reactivity.

NIH Research Projects · FY 2026 · 2025-01

Abstract The humoral immune response at the intestinal interface has evolved to control commensal communities and prevent entry of pathogenic microbes and toxins. B lymphocytes in the gastrointestinal tract are constantly stimulated by microbial antigens and are geared toward generation of immunoglobulin A (IgA), which is the main antibody isotype found in the intestine. B cells mount a rapid antibody response against bacteria via germinal center reaction, a complex multistep process of B cell proliferation and selection, which leads to the generation of antibody-secreting plasma cells or memory B cells. Central for the germinal center reaction are Follicular Dendritic Cells (FDCs), specialized stromal cells that play a crucial role in displaying antigens to germinal center B cells. While our knowledge of how germinal center B cells develop, survive and function has increased exponentially thanks to the generation of specific genetic model, a detailed, mechanistic map of FDC functions during germinal center reaction in response to intestinal commensals and oral vaccination has not been charted yet, mainly because experimental tools to study FDCs are not available. Several lines of evidence indicate that FDCs play multiple role in organizing B cell response, both via secreted and membrane-bound molecules. In the gut, cells are exposed to multiple environmental cues, so it is conceivable that FDCs might integrate those signals to tune germinal center reaction and IgA response. Yet, the exact mechanistic contribution of FDCs in mounting IgA response remains elusive. In this application, we will generate a novel, inducible Cre mouse to specifically target FDCs. This approach relies on a binary Cre approach therefore increasing specificity. We will test the hypothesis that FDCs are essential for maintenance of IgA titer and continuous humoral response to commensal, toxins and enteric pathogens. We also hypothesize that intracellular and extracellular receptors signaling in FDC is essential to coordinate germinal center reaction and tune proper generation of memory B cells and plasma cells. Our aims are: 1) to develop a FDC inducible split-Cre model, and 2) to dissect metabolic and molecular sensing by FDCs in the intestine. The proposed studies will rigorously examine the role of FDCs on generation and maintenance of intestinal humoral immunity. It is our expectation that these studies will increase our understanding of how intestinal antigen display shapes B cell response and adaptive immunity. Furthermore, these studies will provide a transformative tools for better understanding of the relationship between FDCs and B cell response in the gut across time and states.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Structural mechanisms of multimeric ATPases The Kelch Lab studies how multimeric ATPases drive the mechanics of biology. These machines facilitate numerous biological pathways, ranging from DNA replication to virus assembly. Despite their strong structural conservation, the mechanisms of multimeric ATPases are varied, with some functioning as processive motors and others as molecular switches. How this divergence in mechanism arises from conserved components remains unknown. Here we investigate the structures and mechanisms of two similar ATPase machines that demonstrate either processive or switch-like functions. The terminase is an exceptionally powerful and processive motor that pumps DNA into viral capsids. Clamp loaders are ‘one-and- done’ (non-processive) protein remodeling switches that open the sliding clamp ring and place it onto DNA for replication and repair. Both of these machines are related pentameric ATPases that assemble into ring-like structures, so the stark differences in function are not easily explained by subunit stoichiometry or overall architecture. The Kelch Lab has been at the forefront of elucidating the structural mechanisms of both clamp loaders and terminases. We determined the molecular organization of the terminase motor, which became the foundation for the past decade in the field. Our structures of clamp loaders in action have provided unprecedented insight into the mechanism of the most conserved components of the replication fork. In both terminases and clamp loaders, transitions between spiral and planar conformations of the ATPase domains have been implicated, indicating that motor or switch mechanisms are driven by similar structural changes. Not only have our studies been impactful in their individual fields but, by comparing these two mechanisms, we have laid the groundwork for understanding divergent ATPase mechanisms. Our previous studies indicate that the differences in mechanism cannot be easily explained by stoichiometry, overall architecture, or conformational changes of the ATPase domains, which implicates timing and coordination of ATPase activity in determining function. Thus, we have identified three open questions that are a prerequisite for understanding how Nature evolved distinct ATPase functions: - How do ATPase machines enforce directionality of the reaction? - How do ATPases coordinate non-ATPase activities? - How do exogenous factors modulate machine function? To address these questions, we have developed a research program that combines structural studies with incisive examination of biochemical activity and biological function. Furthermore, we have assembled a team of collaborators whose expertise complements that of the Kelch Lab to maximize the scope, breadth, and impact of our work. Our proposed studies will reveal the mechanisms of two evolutionarily ancient and important machines, which will not only impact the fields of DNA replication/repair and virus assembly but also ATPase biology in general. There are numerous diseases associated with dysregulation of sliding clamps and clamp loaders, and terminases are the target of FDA-approved drugs against viral pathogens. Therefore, our studies have the potential to impact human health. More broadly, our studies lay the groundwork for engineering ATPase nanomachines with novel activities or modes of regulation.

NIH Research Projects · FY 2026 · 2025-01

SUMMARY. The primary objective of this K23 award is to provide Dr. Nisha Fahey, DO, MSc, Assistant Professor in the Department of Pediatrics at the UMass Chan Medical School, with the research training, mentorship, and protected time necessary to accelerate her career as an independent physician-scientist committed to improving the health of mother-infant dyads. As a primary care pediatrician with global health fellowship training, Dr. Fahey is committed to reducing infant mortality among low birth weight infants, particularly in global settings. Her K23 research will focus on promoting the practice of Kangaroo Care, featuring skin-to-skin care, among mothers of low birth weight infants in rural India. Despite the lifesaving benefits of this low-cost therapy, community-based Kangaroo Care for low birth weight infants discharged from the Neonatal Intensive Care Unit (NICU) remains a crucial gap in research to prevent neonatal mortality. This proposal seeks to develop a multicomponent intervention that promotes the practice of Kangaroo Care after NICU discharge among mothers of low birth weight infants in rural India. The effects of poverty and adverse social circumstances further increase their risk of mortality. To achieve this goal, Dr. Fahey seeks to develop expertise in specific methodology and content areas including community-engaged research, implementation science, and trial design, including multiphase optimization strategy (MOST) design, in global settings. If granted this K23 award, Dr. Fahey will use these methods to perform a MOST design-based trial centered on the premise that the continuation of Kangaroo Care after NICU discharge among the mothers of low birth weight infants in rural Indian communities requires: 1) engaging communities to refine and pilot test community-informed interventions (Aims 1 and 2) and 2) developing an optimized, multicomponent intervention that balances impact and resources (Aim 3). Dr. Fahey leads a ten-year institutional collaboration between Bhaikaka University and UMass Chan Medical School focused on capacity-building research and community health in rural India, which ideally positions her to perform this proposed study. By the conclusion of this award, Dr. Fahey’s research and training plan will yield a community-informed, optimized, multicomponent intervention and the skillset to conduct an effectiveness trial in an R01 (PAR-22-105). Dr. Fahey has a well-established team of mentors with clinical and research expertise along with the track record and commitment needed to successfully support her in establishing a federally funded independent program of research to enhance maternal and infant health in global settings. After thoughtful review of the critiques received, Dr. Fahey and her mentorship team have carefully crafted a training plan that builds on existing strengths and addresses current gaps. This K23 award will build on existing international community partnerships and equip Dr. Fahey with the skills to advance her emerging program of research to focus on intervention development and evaluation in global settings.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT The development of human type 1 diabetes (T1D) involves complex interactions between pancreatic β-cells and the immune system that are still not fully understood. We propose to study the dynamic interactions between human immune systems from individuals with T1D and autologous human islets derived from donor iPSC (SC- islets) using translational humanized mouse models and in vitro metabolic profiling platforms. This proposal is in response to RFA-DK-23-004 for Human Islet Research Network - Consortium on Modeling Autoimmune Diabetes (HIRN-CMAD) (UG3/UH3) that has a primary goal to support “the development of in vitro and in vivo models of T1D to enable studies of human T1D pathophysiology and to serve as platforms for preclinical assessments of new T1D interventions.” Our long-term goal is to generate humanized mice to model T1D using human SC-islets differentiated from T1D iPSC and co-engrafting autologous immune systems from primary PBMC or SC-derived hematopoietic cells. The proposed models would enable the direct study of interactions between human immune cells and autologous SC-islets for both effector mechanisms and the exchange of metabolites regulating inflammation. A central feature for our application is the cutting-edge humanized mouse models developed by Dr. Leonard Shultz at The Jackson Laboratory. Recent advancement in the NOD-scid IL2rgnull (NSG) strains by the Shultz lab have created new models that 1) support the engraftment of human B cells, NK cells and T cells following PBMC injection, 2) bear the W41 mutation in the mouse kit gene and are more permissive to HSC engraftment in the absence of irradiation preconditioning and 3) enable the generation of peripheral lymph nodes, including pancreatic lymph nodes, after engraftment with umbilical cord blood (UCB)- CD34+ HSC when treated with an AAV vector expressing mouse thymic stromal lymphopoietin (TSLP). We have assembled a team of investigators from 4 institutions, UMCMS, Harvard, Joslin and The Jackson Laboratory to test our overall hypothesis that our proposed studies of autologous human SC-islets and immune cells transplanted into novel humanized mouse models accompanied by in vitro metabolic profiling will increase the understanding of dynamic cellular and metabolic interactions that drive human T1D. We will test this hypothesis in 3 Aims; Aim 1. Interrogate interactions between autologous immune systems and SC-islets in vivo, Aim 2. Identify and characterize metabolic pathways influencing SC-islet survival during inflammation, and Aim 3. Develop SC-HSC from iPSC and explore the role of pancreatic lymph nodes in T1D. The proposed studies will provide a mechanistic understanding of the factors that contribute to the induction of T1D in humans and will provide critical benchmarks for the effective design of therapeutic strategies for T1D and for the identification of specific patient populations that will respond optimally to treatment modalities.

NIH Research Projects · FY 2025 · 2025-01

PROJECT SUMMARY Workforce diversity is an urgent priority for the National Institutes of Health (NIH). However, there are few initiatives to diversify the autism research workforce. The problem of workforce diversity in research has far- reaching clinical implications. A workforce that represents the community that it serves can address cultural beliefs and practices, as well as stigma, that may impact autism diagnosis and treatment, and ultimately lead to better quality of care. Given the relation between workforce diversity and research/clinical outcomes, the objective of this proposal, “Building a Diverse Workforce in Autism Research: From Community Partnerships to Policy” is to increase diversity in the autism research workforce by convening a conference focused on career advancement for individuals who are underrepresented in the biomedical research workforce. This conference was previously hosted in 2023 with enormous success, and this will be the second year of this conference in 2024. We propose three specific aims focused on providing networking opportunities, didactic and applied training activities, and research experiences. The proposed conference is relevant to the scientific mission of the National Institute of Mental Health (NIMH) because it will provide strategic and targeted career advancement opportunities (short-term goal) that will pave the way for the next generation of researchers underrepresented in biomedical research (long-term goal). Through creativity and innovation, diversity in the workforce has the potential to transform the treatment of autism, by increasing access to evidence-based treatments, fostering high-quality care, and improving clinical outcomes, particularly for underrepresented communities. The objective of this conference is aligned with NIMH’s strategic plan (Goal 4) to strengthen the public health impact of research by linking junior and senior investigators on a wide range topics, from community partnerships to policy. Our proposal is innovative because we will launch the first conference designed to support underrepresented students, post-doctoral fellows, and junior faculty in biomedical research across the translational pipeline.

NIH Research Projects · FY 2026 · 2024-12

PROJECT ABSTRACT Immune dysregulation is a hallmark of severe COVID-19 caused by infection with severe acute respiratory syndrome coronavirus 2 (SCoV2). The pathogenesis of immune dysregulation in COVID-19 is thought to involve a dysfunctional host innate immune response, including a discordant type I interferon (T1IFN) response relative to viral replication and an exaggerated inflammatory response creating an imbalance between viral clearance and excessive inflammation ultimately resulting in acute lung injury (ALI), multiorgan failure, and even death. Despite extensive investigation, the precise mechanisms underlying the immune dysfunction during SCoV2 infection remain a mystery. We recently discovered a novel innate immune pathway mediated by A Disintegrin and Metalloproteinase 9 (ADAM9) critical for the host response to positive, single-stranded RNA (+ssRNA) viruses, including SCoV2. Specifically, we demonstrated that ADAM9 is essential for melanoma differentiation- associated protein 5 (MDA5)-mediated T1IFN response to cytosolic SCoV2 viral RNA (vRNA). Our preliminary results in SCoV2-infected mice revealed a profound difference in clinical disease severity based on ADAM9 expression alone, suggesting that ADAM9 is associated with the development of severe disease, consistent with published data; however, the mechanisms by which ADAM9 drives disease pathology are unknown. The current proposal builds logically on our preliminary data that revealed a critical role for ADAM9 in MDA5-mediated innate immune activation and aims to define the role of ADAM9 in the immunopathogenesis of severe COVID-19 and determine the impact of ADAM9 on SCoV2-induced acute lung injury. Successful completion of these studies will provide further insight into the novel ADAM9/MDA5 innate immune pathway and the role of this pathway in the pathogenesis of SCoV2-mediated immune dysregulation and its impact on lung pathology. This project builds logically on the candidate’s previous postdoctoral research and new compelling preliminary data and will serve as a career development mechanism. The candidate will receive mentorship from Dr. Fitzgerald and Dr. Kurt-Jones on experimental design, data interpretation, grant and manuscript preparation, lab protocol development, training/mentoring of graduate students, seminar preparation and presentation skills, and additional support to ensure a seamless transition to an independent investigator. Data generated from this proposal will lay the foundation for an R01 that builds upon the current study to further investigate virus-host interactions regulating innate immune mechanisms and determine immune correlates of protection versus pathogenesis that can be leveraged to develop novel strategies to treat or prevent lethal disease. These studies will be conducted at the UMass Chan Medical School, a leading institution for research in innate immunity and viral pathogenesis.

NIH Research Projects · FY 2026 · 2024-12

New treatments are urgently needed for parasitic gastrointestinal nematodes (GINs; hookworms, whipworms, roundworms) that infect ~1.5 billion people and cause significant morbidity in children, pregnant women, and working adults. The main drug used against GIN in mass drug administration (MDA) is albendazole (ABZ), with increased use of ivermectin (IVM) and consideration of the experimental drug emodepside (EMO). MDA with ABZ has not been curative, and IVM and EMO also have limitations. Thus, controlling GINs remains a critical unmet medical need. Experience with HIV, tuberculosis, and malaria shows that well-designed drug combinations can suppress resistance and enhance efficacy. Especially effective combinations are synergistic and show collateral sensitivity (CS), where resistance to drug A increases susceptibility to drug B. We have pioneered the development of crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) as safe, effective, and affordable next-generation anthelmintics to control GINs. For over 60 years, Cry proteins have controlled insects that vector disease and damage crops, including use in >100 million hectares of transgenic crops grown annually. Oral Cry5Ba treatment is safe for mammals and highly effective against GINs in rodents, dogs, sheep, pigs, and horses. We have now identified five other anthelmintic Cry proteins. Cry5Ba interacts synergistically and shows CS with nicotinic acetylcholine receptor agonist anthelmintics. Cry5Ba also synergizes with ABZ against all GINs and shows CS to EMO- and IVM-resistant nematodes. These data demonstrate that Cry proteins have excellent combinatorial properties with small molecule anthelmintic drugs. We propose to discover optimal combinations between six anthelmintic Cry proteins and three small molecule drugs (ABZ, IVM, or EMO) to suppress drug resistance and greatly improve efficacy. Three major aims will be pursued: Aim 1. Find strong synergistic interactions. Checkerboard design studies will be carried out with 6 Cry proteins x 3 small molecule drugs against three major GINs: Ancylostoma ceylanicum hookworms, Trichuris muris whipworms, and Ascaris suum roundworms. Synergy will be quantified with the ZIP synergy model. Aim 2. Find strong CS relationships. (1) Quantify the sensitivity of ABZ, IVM, and EMO-resistant Caenorhabditis elegans to six Cry proteins and the sensitivity of Cry-resistant C. elegans to ABZ, IVM, and EMO. (2) Quantify the sensitivity of ABZ- and IVM-resistant Haemonchus contortus to (a) Cry proteins and (b) Cry protein – small molecule drug combinations using a larval development assay. The data from Aims 1+2 will be integrated to design and test optimal Cry protein-drug combinations for in vivo synergy against parasitic infections, mechanistic studies using transcriptomics, and resistance management via computer modeling to optimize strategies for sustaining efficacy against GINs. The results will identify optimal combinations of Cry proteins and small molecule drugs to suppress drug resistance and increase efficacy, thereby providing durable relief from the human suffering caused by GINs.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Synthetic messenger RNAs (mRNAs) represent a new class of biopharmaceuticals with broad clinical utility for a range of diseases. The incorporation of chemically modified uridine nucleosides, pseudouridine (Ψ) and N1- methylpseudouridine (m1Ψ), significantly reduces synthetic mRNA immunogenicity. Linear nucleoside-modified mRNAs are short-lived because they are susceptible to cellular exonucleases, hindering their broad clinical utility for a range of diseases. Synthetic circular mRNAs (circRNAs) evade cellular exonucleases, resulting in a longer half-life, but efficient circularization methods are incompatible with chemical modifications. The most efficient method of RNA circularization utilizes self-splicing group I introns. Proper folding—and thus activation—of the typical group I intron is disrupted by Ψ-modified nucleotides. A survey of self-splicing introns, however, identified a compact group I intron from the cyanobacterium Azoarcus that effectively circularizes short (~150 nt) Ψ- modified RNA. The Azoarcus intron can circularize long (~2000 nt) unmodified mRNA but not long Ψ-modified RNAs. This proposal seeks to test the hypothesis that Ψ and m1Ψ prevent circularization by disrupting tertiary interactions required for rapid and efficient intron splicing and to develop efficient circularization techniques compatible with Ψ and m1Ψ for safe and stable RNA therapeutics. Aim 1 will determine how uridine modifications affect the structure of Azoarcus group I intron. High throughput structural probing will be used to study how uridine modifications stabilize inactive conformations of the Azoarcus group I intron. Aim 2 will employ in vitro evolution to identify Azoarcus group I intron variants optimized to circularize Ψ and m1Ψ-modified RNA. The Ψ- and m1Ψ-modified circRNA will be tested in immune cells for their ability to induce an immune response. This study will provide structural insights into how nucleoside modifications change RNA structure and function and develop a straightforward methodology to prepare nucleoside-modified circular mRNAs, expanding the potential uses of mRNA therapeutics beyond vaccines. In addition, the proposed research will provide training in high- throughput sequencing, in vitro biochemistry, cellular RNA sensing, nucleic acid chemistry, and structural biology to prepare the fellow for a career as an independent investigator developing next-generation mRNA therapeutics.

NIH Research Projects · FY 2026 · 2024-12

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative motor neuron disease that is largely untreatable and leads to death within 5 years of diagnosis. ~10% of ALS cases are familial and caused by mutations in various ALS genes. Ultimately, the ideal treatment for genetic diseases such as ALS is somatic gene correction. Recently, advances in CRISPR/Cas systems have shown considerable promise for precise editing of disease loci using base and prime editing systems delivered by AAV. The second-most prevalent cause of familial ALS are mutations in the SOD1 gene. These mutations confer multiple toxic properties onto the protein. This project proposes to develop treatment to achieve somatic gene correction for common missense mutations in SOD1. The aims of this proposal are: (1) To develop AAV-mediated base editing gene correction strategies for the SOD1 A5V mutation in vitro. We will create next-generation base editors with a compact size, increased efficiency, and greater control over bystander editing. (2) To develop AAV-mediated prime editing gene correction strategies for the SOD1 A5V and G94A mutations in vitro. Different prime editor systems will be tested for optimal editing efficiencies and low off-target editing. (3) In in vivo studies, examine and optimize AAV- mediated base and prime editing gene correction strategies for the A5V and G94A mutations in A5V and SOD1G93A mouse models. Mice will receive AAV-mediated base and prime editors through an intracerebroventricular injection. Base and prime editor strategies will first be screened in mutation carrying HEK293T cells and then optimized in patient fibroblasts and mouse models. The effects of gene correction on gain- and loss-of-function molecular and motor phenotypes will next be evaluated. The fundamental hypothesis driving this proposal is that AAV-mediated somatic gene correction strategies, using base editing or prime editing to target the SOD1 mutations A5V and G94A, will decrease toxic GOF pathology and increase WT SOD1 protein levels in vivo, resulting in a balanced treatment for SOD1-ALS and a rescue of motor phenotype. In addition, with mentorship from experts in ALS and gene editing and the wealth of resources available at UMASS Chan, these studies will provide extensive training in gene editing for CNS diseases and project development that will be an essential foundation for a future career as an independent researcher developing gene therapies for a range of genetic CNS diseases.

NIH Research Projects · FY 2026 · 2024-11

PROJECT SUMMARY Gene therapies based on adeno-associated virus (AAV) vectors have been a revolutionary medical advancement in treating human genetic diseases. With a single dose, AAV-mediated gene therapies can confer long-term correction or abatement of disease for the lifetime of the patient. Recombinant (r)AAVs that are used for the transfer of therapeutic genes are considered safe, partly because these vectors are removed of all viral genes. The only viral elements that are retained are the inverted terminal repeat (ITR) sequences that are at both the ends of the vector genome. However, this feature may make them vulnerable to natural viral infections from wildtype (wt)AAV and helper viruses, such as adenoviruses (AdV), herpesviruses, or papillomaviruses. It is speculated that these challenges may destabilize the therapeutic vector genome or may amplify them in a process called “mobilization”. Unfortunately, models for these events have yet to be explored, and the extent to which these processes can alter the abundance or structure of the vector genomes are unknown. Understanding the stability of rAAV genomes in treated tissues under these naturally occurring and unavoidable circumstances is critically important for the gene therapy field. This project proposal aims to define if and how natural virus infections that can act to alter the composition of AAV-based gene therapy in human hepatocytes, a prevalent gene therapy target for rAAV-based treatments. The objectives are to develop novel methods to characterize the structure of vector genomes as they transition from linear species, to extrachromosomal episomes, and to vector genomes that have integrated into the host cell genome. The bases of this work leverages expertise in long-read sequencing technologies, bioinformatics, and vectorology to address these decades-long unanswered questions. The grant proposal is divided into two main aims: • Aim 1. Track changes in vector genome abundance and episomal configurations within transduced primary human hepatocytes following wtAAV and wtAdV infection. A relevant in vitro model will be developed to track the kinetics of transgene expression and to assess the stability of rAAV genomes in transduced human hepatocytes following wtAAV and wtAdV infection over time. • Aim 2. Assessing the ability for wtAAV and wtAdV infection to destabilize rAAV episomes in humanized mouse livers. Whether wtAAV and wtAdV infection will act to destabilize or mobilize rAAV genomes in human hepatocytes, or whether they will act to drive integration of vectors into the host cell genome will be tested in the Fah/Rag2/Il2rgc-deficient (FRG)-humanized liver mouse model.